Proteins are relatively unstable in the aqueous state and undergo chemical and physical degradation resulting in a loss of biological activity during processing and storage (Manning, et al. (1989), Pharm. Res., 6:903-918). The process of drying (e.g., freeze-drying, spray drying and air drying) is often employed to stabilize proteins for long-term storage, particularly when the protein is relatively unstable in liquid formulations.
A lyophilization cycle is usually composed of three steps: freezing, primary drying and secondary drying (Williams and Polli (1984), J. Parenteral Sci. Technol. 38:48-59). In the freezing step, the protein solution is cooled until it is adequately frozen. Bulk water in the protein solution forms ice at this stage. This ice sublimes in the primary drying stage which is conducted by reducing chamber pressure below the vapor pressure of the ice using a vacuum. Finally, sorbed or bound water is removed at the secondary drying stage under reduced chamber pressure and elevated shelf temperature. The process produces a material known as a lyophilized cake. Thereafter the cake is reconstituted. The standard practice is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in production of pharmaceuticals for parenteral administration (Chen, Drug Dev. Ind. Pharm., 18:1311-1354 (1992)).
Spray-drying is typically achieved by microdispersing a solution into a stream of hot gas. The solution is continously fed into the gas stream and microdispersion (atomization) is achieved through use of a pressurized air stream (Masters, in "Spray-Drying Handbook" (5th ed.) Longman Scientific and Technical, Essez, U.K., (1991), pp. 491-676). When applied to proteins, the temperature of the gas stream typically ranges from 80.degree.-200.degree. C. (Broadhead et al. (1992), Drug Devel. Ind. Pharm., 18:1169-1206; and Mumenthaler et al. (1994), Pharm. Res., 11:12-20).
Air-drying is typically performed by placing solutions at ambient temperature in a very low humidity environment. The solutions are continuously exposed to the low humidity air until they are sufficiently dry (Carpenter and Crowe (1988), Cryobiology, 25:459-470; and Roser (1991), Biopharm, 4:47-53).
Dried proteins are subject to conformational instability induced by the acute stresses encountered during drying. Protein stability during drying is a function of environmental factors which include temperature, humidity, pH, ionic strength, and solvent medium composition. Even when the protein survives drying without significant damage, damage may occur during storage of the dried product (Pikal, BioPharm, 27:26-30 (1990)). Damage to dried proteins is manifested after rehydration, for example, as a loss of protein solubility, aggregation, loss of activity in appropriate bioassays or in the case of enzymes, a loss of catalytic activity (Carpenter et al. (1991) Develop. Biol. Standard., 74:225-239; WO 93/00807, to Carpenter; Broadhead et al. (1992), supra; Mumenthaler et al. (1994), supra; Carpenter and Crowe (1988), supra; and Roser (1991), supra).
Typical practices to improve protein stability are addressed by varying the formulation. For example, excipients are added to the protein solution or suspension prior to drying to improve the stability of the protein to the drying process, and to improve the storage stability of the dried product. (Carpenter et al. (1991), supra; and Pikal, (1990), supra). Commonly used excipients include sugars (e.g., sucrose, glucose, lactose, trehalose); amino acids (e.g., glycine, alanine, serine, proline, sodium glutamate, lysine, aminobutyric acid); proteins (e.g., human serum albumin, bovine serum albumin); glycerol; polyols (e.g., xylitol, mannitol, inositol, sorbitol); amines (e.g., betaine, sarcosine, trimethylamine N-oxide); salts (e.g., hydrogen chloride, phosphates, sodium acetate, magnesium sulfate, sodium chloride, ammonium sulfate and sodium sulfate); ethylene glycol; polyethylene glycol; 2-methyl-2,4-pentanediol and dimethylsulfoxide (Arakawa et al. (1990), Cryobiology, 27:401-415; Carpenter et al. (1991) supra; Pikal, (1990), supra; Broadhead et al. (1992), supra; Mumenthaler et al. (1994), supra; Carpenter and Crowe (1988), supra; and Roser (1991), supra).
While the use of additives has improved the stability of dried proteins, many proteins which are subject to drying and subsequent storage contain unacceptable or undesirable amounts of inactive, aggregated protein in the rehydrated formulation (Townsend and DeLuca (1983), J. Pharm. Sci., 80:63-66; Hora et al. (1992), Pharm. Res., 9:33-36; Yoshiaka et al. (1993), Pharm. Res., 10:687-691; Izutsu et al. (1991), Int. J. Pharm., 71:137-146; Liu et al. (1991), Biotechnol. Bioeng., 37:177-184; Pikal et al. (1991), Pharm. Res., 8:427-436; Townsend et al. (1990), Pharm. Res., 7:1086-1090; Broadhead et al. (1992), supra; Mumenthaler et al. (1994), supra; Carpenter and Crowe (1988), supra; and Roser (1991), supra). This is particularly problematic when preparing pharmaceutical formulations, because aggregated proteins have been known to be immunogenic (Cleland et al. (1993), Crit. Rev. Therapeutic Drug Carrier Systems, 10:307-377; Robbins et al. (1987), Diabetes, 36:838-845; and Pinckard et al. (1967), Clin. Exp. Immunol., 2:331-340).
Thus, there is a need to develop further means for increasing the amount of active protein from reconstituted formulations of dried proteins. It is to this and other objects that the present invention is directed.